Phasmid species that inhabit colder environments are less likely to have the ability to fly

Abstract A vast majority of insects can fly, but some cannot. Flight generally increases how far an individual can travel to access mates, enables the exploitation of additional food resources, and aids in predator avoidance. Despite its functional significance, much remains unknown about the factors that influence the evolution of flight. Here, I use phylogenetic comparative methods to investigate whether average annual temperature or wind speed, two components of the flying environment, is correlated with the evolution of flight using data from 107 species of stick and leaf insects (Insecta: Phasmatodea). I find no association between wind speed and flying ability in this clade. However, I find that colder temperatures are associated with the lack of flying ability. This pattern may be explained by the additional metabolic costs required for insects to fly when it is cold. This finding contradicts previous patterns observed in other insect groups and supports the hypothesis that cold temperatures can influence the evolution of flight.

Appendix S1. Cold temperature is associated with the lack of flying ability in stick insects, even when taking body size into consideration. I investigated the cold temperature and high windspeed hypotheses using another phasmid phylogeny that had corresponding data on flying ability (Zeng et al. 2020). This dataset also included information on body size, which allowed me to investigate whether the patterns observed and reported in the main text would be similar when including body size as a covariate.

DATA COLLECTION
The phylogeny used in Zeng et al. (2020) includes 129 phasmid taxa, with representatives from ~70% of the phasmid tribes. This time-calibrated Bayesian Inference phylogeny is based on a BEAST (Drummond et al. 2012) analysis using 4 loci (2 nuclear and 2 mitochondrial). Flying ability was determined by using wing and body size data collected from photographs and the literature (Zeng et al. 2020). Species without wings were considered incapable of flight. Winged taxa had a bimodal distribution for relative wing size, and species with wings that were 40% as long as their body size were considered to have long wings and assumed to be able to fly. Species with long wings were coded as "1", while all others were coded as "0" (Dataset S4). Since male body size was going to be used in the analyses, only male wing size was considered when determining whether a species was considered capable of flight.
rGBIF was then used to query the global biodiversity information facility (i.e. GBIF) for occurrence data for all 129 taxa on July 14, 2022 (GBIF Dataset). This resulted in 11,449 raw occurrences (GBIF Dataset). Mean monthly temperature and windspeed data (at a resolution of ~20km 2 ) was then extracted for each phasmid occurrence point using the r package raster (version 3.5; Hijmans 2022). To get yearly average temperature and windspeed data, the monthly averages were summed and divided by 12. Duplicated data points and those for which temperature and windspeed could not be acquired were then removed from the dataset. This step reduced the number of occurrences to 9,732 (Dataset S5).
Previous studies (e.g. Howard et al. 2019), and our main analyses, remove species with less than 3 unique occurrence points. However, in this case, this decision would result in only 29 species being included in the final dataset (and only 12 of which had long wings). Thus, I reduced the number of occurrence points required to 1, which resulted in 40 species being included in the final dataset. Outgroup taxa were not included in this dataset.

STATISTICAL ANALYSES
To test the hypotheses that windspeed and temperature influence the evolution of flight in stick insects I conducted multiple phylogenetic logistic regressions (Ives and Garland 2010) using phylolm (version 2.6.2; Ho and Ané 2014) in R (version 4.2.1; R Core Team 2022). First, I investigated whether an environmental variable (i.e. temperature or windspeed), body size, and their interaction influence the evolution of flight. For these analyses, I used p > 0.15 to identify whether the interactions were insignificant (to be more conservative, following Bursac et al. 2008), which they were. Thus, I conducted two additional phylogenetic logistic regressions in which the interaction term was not included (i.e. the interactions were dropped from the models).

Results
Using the Zeng et al. (2020) dataset, I found that windspeed was not significantly associated with flying ability (estimated coefficient=-0.803, z=-1.693, p=0.090). However, I found that colder temperatures were associated with the inability to fly (estimated coefficient=0.176, z=2.502, p=0.012; Figure S2). Thus, these results (which correct for body size) are concordant with the main results despite (1) the much smaller sample size, (2) the inclusion of different taxa, and (3) a different phylogenetic hypothesis. Additionally, in both models, I found that larger body size is associated with the evolution of flight in phasmids (as previously identified in Zeng et al. 2020; windspeed model: estimated body size coefficient=0.051, z=2.891, p=0.004; temperature model: estimated body size coefficient=0.074, z=3.308, p=0.001; Figure S2).